EP1509767A1

EP1509767A1 - Detection of autoantibodies to cytokeratin 18 protein in patients with bronchial asthma and chronic rhinitis, and its applications including a kit for diagnosing bronchial asthma and chronic rhinitis comprising mammalian cytokeratin 18 protein

Info

Publication number: EP1509767A1
Application number: EP03723443A
Authority: EP
Inventors: Dong-Ho Nahm; Sook-Yeong Jeon
Original assignee: Individual
Current assignee: Individual
Priority date: 2002-05-15
Filing date: 2003-05-12
Publication date: 2005-03-02
Also published as: AU2003230328A1; KR20030089530A; US20070231842A1; JP2005531754A; US20050208583A1; WO2003098211A1; KR100495241B1

Abstract

The present invention is based on the surprising discovery of autoantibodies to cytokeratin 18 protein in the serum samples of patients with bronchial asthma and chronic rhinitis, especially in nonallergic patients. The present invention includes diagnostic methods and a diagnostic kit to detect patients with bronchial asthma and chronic rhinitis associated with autoantibodies to cytokeratin 18. The invention also includes methods and kits to prescribe or monitor treatment for patients with bronchial asthma and chronic rhinitis by detecting autoantibodies to cytokeratin 18. The present invention also includes a pharmaceutical formulation comprising cytokeratin 18 protein to protect the patients with bronchial asthma and chronic rhinitis associated with autoantibodies to cytokeratin 18. The present invention also includes methods to treat the patients with bronchial asthma and chronic rhinitis associated with autoantibodies to cytokeratin 18 using compounds that inhibit the interaction between such autoantibodies and cytokeratin 18 protein.

Description

[Specification]

[Title of the Invention]

Detection of autoantibodies to cytokeratin 18 protein in patients with bronchial asthma and chronic rhinitis, and its applications including a kit for diagnosing bronchial asthma and chronic rhinitis comprising mammalian cytokeratin 18 protein

[Technical Field of the Invention]

The present invention relates to diagnostic methods and a diagnostic kit to detect patients with bronchial asthma and chronic rhinitis associated with autoantibodies to cytokeratin 18.

The present invention includes a pharmaceutical formulation comprising cytokeratin 18 protein to protect or treat the patients with bronchial asthma and chronic rhinitis associated with autoantibodies to cytokeratin 18. The present invention also includes methods to protect or treat the patients with bronchial asthma and chronic rhinitis associated with autoantibodies to cytokeratin 18 using compounds that inhibit the interaction between such autoantibodies and cytokeratin 18 protein.

[Description of Related Art and Background of the Invention] Definition and prevalence of bronchial asthma and chronic rhinitis

Bronchial asthma is defined as a chronic inflammatory disease of the airways characterized by exacerbations of coughing, wheezing, and difficult breathing that are usually reversible but can be severe and sometimes fatal (National Asthma Education and Prevention Program. NIH publication No. 97-4051, 1997). Bronchial asthma is a common disease, affecting about 5 to 10% of the population in developed countries. Additionally, the prevalence of bronchial asthma is increased over recent decades probably due to environmental factors (Sears MR. Lancet 1997; 350(Suppl2): 1-4.).

Chronic rhinitis is defined as an inflammatory disease of nasal airway characterized by typical chronic symptoms of rhinorrhea, sneezing, and nasal obstruction (Dykewicz MS et al., Ann Allergy Asthma Immunol 1998; 81 :463-468.). Chronic rhinitis is also very common and affects about 10 to 20% of the population in developed countries (Sly RM., Ann Allergy Asthma Immunol 1999; 82:233-48).

Bronchial asthma and chronic rhinitis are linked by epidemiologic and immunopathologic characteristics and common therapeutic approaches. Most patients with bronchial asthma (about 80-99%) also have chronic rhinitis, and about 30-40% of patients with chronic rhinitis also have bronchial asthma as well (Vignola AM et al., Clin Exp Immunol 2001; 31:674-677; Simons FE., J Allergy Clin Immunol 1999; 104:534- 540). The same histological type of respiratory epithelium lines the upper and lower airways. In pathological view point, patients with chronic rhinitis have similar features of inflammation in the upper airway (nasal mucosa tissue) comparable to those in the lower airway (bronchial mucosa tissue) of patients with bronchial asthma. Recently the new term "rhinobronchitis" was suggested to facilitate appropriate recognition and treatment of the common inflammatory process throughout the upper (rhinitis) and lower airways (asthma) (Simon FE., J Allergy Clin Immunol 1999; 104:534-540). 'Aspirin-exacerbated respiratory disease' is a clinical syndrome characterized by the presence of chronic rhinitis, nasal polyps, asthma, and precipitation of both asthma and rhinitis attacks after ingestion of aspirin (Berges-Gimene MP et al., Ann Allergy Asthma Immunol 2002; 89:474-478). The existence of above syndrome provides evidence that a common pathogenetic mechanism works in both bronchial asthma and chronic rhinitis (Picado C, Curr Allergy Asthma Rep 2002; 2:488-493).

Etiology and pathogenetic mechanism of bronchial asthma and chronic rhinitis

The primary etiology and mechanism causing the development of bronchial asthma and chronic rhinitis is not completely understood yet (National Asthma Education and Prevention Program. NIH publication No. 97-4051, 1997; Dykewicz MS et al., Ann Allergy Asthma Immunol 1998; 81 :463-468). Traditionally, allergic immune response to common environmental agents (allergens such as house dust mite and pollens) has been regarded as an important mechanism responsible for the development of airway inflammation in patients with bronchial asthma and chronic rhinitis (Lemanske RF Jr et al., JAMA 1997; 278:1855-1873; Dykewicz MS et al., Ann Allergy Asthma Immunol 1998; 81:463-468.). However, allergic response to common environmental agents cannot be detected in significant portions (about 40-50%) of patients with bronchial asthma and chronic rhinitis (Pearce N et al., Thorax 1999; 54:268-272; Settipane RA et al., Ann Allergy Asthma Immunol 2001;86:494-508). These patients have been classified as having nonallergic asthma and rhinitis. Nonallergic asthma and rhinitis are often begins in an older age and is clinically more severe than allergic asthma and rhinitis (Virchow JC Jr. et al., J Allergy Clin Immunol 1996;98:S27-S33; Settipane RA et al., Ann Allergy Asthma Immunol 2001 ;86:494-508). However, the mechanism responsible for the development of airway inflammation in patients with nonallergic asthma and rhinitis cannot be explained yet. Diagnosis, classification, and treatment of bronchial asthma and chronic rhinitis The diagnosis of bronchial asthma and chronic rhinitis can be achieved by a characteristic history and objective tests (National Asthma Education and Prevention Program. NIH publication No. 97-4051, 1997; Dykewicz MS et al., Ann Allergy Asthma Immunol 1998;81:463-468).

Bronchial asthma can be diagnosed when history of typical clinical symptoms such as intermittent cough, dyspnea, or wheezing is present and reversibility of airway obstruction can be documented by pulmonary function measurements before and after inhalation of bronchodilator. Demonstration of airway hyper-reactivity to nonspecific stimuli (methacholine or histamine, etc.) also can be an objective evidence for the diagnosis of bronchial asthma.

Chronic rhinitis is mainly diagnosed by typical history of chronic symptoms such as rhinorrhea, sneezing, and nasal obstruction rather than objective laboratory tests. Demonstration of eosinophilic leukocyte in nasal secretion or nasal mucosa tissue by microscopic examination also can be sometimes helpful for the diagnosis of chronic rhinitis.

In patients with bronchial asthma and chronic rhinitis, presence of allergic reaction to common environmental inhalant agents (allergens such as house dust mite and pollens) can be examined by allergy skin test or by in-vitro tests for specific IgE antibodies to allergens in serum samples. The examination of allergic reaction to environmental agents is clinically useful for the identification of environmental risk factors, which can precipitate the exacerbation of bronchial asthma and chronic rhinitis and also useful for classification of allergic patients with bronchial asthma and chronic rhinitis from nonallergic patients with such diseases. However the examination of allergic reaction to environmental allergens cannot be used for diagnosis of bronchial asthma and chronic rhinitis because positive reaction is also present in about 20-30% of apparently healthy people and more than 50% of patients with other diseases like atopic dermatitis and allergic conjunctivitis (Pearce N et al., Thorax 1999; 54:268-272.). In allergic patients with bronchial asthma and chronic rhinitis, clinical symptoms can be improved by reducing exposure to sensitized allergens or by reducing the patient's sensitivity to allergens through immunotherapy. During immunotherapy, the allergens are regularly administered hypodermically in order to reduce the allergic reaction to those allergens (National Asthma Education and Prevention Program. NIH publication No. 97- 4051, 1997; Dykewicz MS et al., Ann Allergy Asthma Immunol 1998; 81 :463-468). As a pharmacological therapy, corticosteroid has been known to be the most effective medication for the treatment of bronchial asthma and chronic rhinitis. Direct administration of corticosteroid to the target tissue by nasal spray or inhalation devices is preferred method than the systemic administration to avoid the systemic side effects. For further symptomatic controls of bronchial asthma, the additional treatment with inhaled bronchodilator can be useful. And oral administration of antihistamine can be also useful for reducing the symptoms of chronic rhinitis (National Asthma Education and Prevention Program. NIH publication No. 97-4051, 1997; Dykewicz MS et al., Ann Allergy Asthma Immunol 1998;81 :463-468). The effectiveness of treatment for patients with bronchial asthma and chronic rhinitis can be monitored by history on the changes of clinical symptoms. Serial measurements of objective pulmonary function can be also useful for monitoring the effect of treatment in patients with bronchial asthma.

Problems in current definition and classification of bronchial asthma and chronic rhinitis

Bronchial asthma and chronic rhinitis are not diseases but syndromes including various heterogeneous diseases regarding etiology, pathogenetic mechanism, and natural history (Rackemann FM., J Allergy 1940;11 :147-162; Nirchow JC Jr. et al., J Allergy

Clin Immunol 1996;98:S27-S33; Dykewicz MS et al., Ann Allergy Asthma Immunol 1998;81:478-518; Sobol SE et al., Curr Allergy Asthma Rep 2001;1: 193-201). Etiological classification of bronchial asthma and chronic rhinitis is difficult because the primary etiology of bronchial asthma and chronic rhinitis is not completely understood yet (National Asthma Education and Prevention Program. NIH publication No. 97-4051, 1997; Dykewicz MS et al., Ann Allergy Asthma Immunol 1998;81 :478-518). Current criteria for classification of bronchial asthma and chronic rhinitis are mainly dependent on the examination of allergic reaction to common environmental allergens. And there is no currently available test method for the direct detection of patients with nonallergic asthma and rhinitis except demonstrating the absence of allergic reaction to common environmental allergens (about 10-30 allergens).

Problems in current method for diagnosis of bronchial asthma and chronic rhinitis

Primary-care physicians mainly depend on the clinical history and physical examination for the diagnosis of bronchial asthma and chronic rhinitis'. The objective laboratory tests are not widely used due to following reasons and this sometimes results in misdiagnosis or delayed diagnosis of such diseases. Pulmonary function measurement needs of special equipment and a trained skilled person. Allergy skin test is accompanied by minor physical discomfort of patients due to needle-prick of skin and needs a skilled person for test. In-vitro test for specific IgE antibodies to common allergens also needs special laboratory equipments and a skilled person and usually need tests for multiple allergens. Examination of nasal eosinophilic leukocyte is not routinely used due to lack of consensus on the diagnostic value of this test (Dykewicz MS et al., Ann Allergy Asthma Immunol 1998; 81 :463-468). And there is no available laboratory diagnostic test for chronic rhinitis with consensus on its diagnostic value.

Pathogenesis and diagnostic methods of other chronic inflammatory diseases

In various kinds of chronic inflammatory diseases such as rheumatoid arthritis, systemic lupus erythematosus, chronic atrophic gastritis, and thyroiditis, the autoimmune response against the self-antigen (autoantigen) is known to play an important role in the pathogenesis of disease. In these diseases, tests for detecting autoantibodies to various autoantigens are widely used for diagnosing and monitoring the diseases, prediction of prognosis, and choice of treatment methods (Davidson A et al., N Engl J Med 2001; 345:340-350).

Problems in the application of autoantibody test for diagnosis and classification of bronchial asthma and chronic rhinitis

Higher incidences of various autoantibodies against antigens in bronchial mucosa, nasal mucosa, paranasal sinus, lung, and endothelial cell have been reported in patients with bronchial asthma and chronic rhinitis, especially in nonallergic patients compared to healthy controls (Girard JP et al., Poumon Coeur 1973; 29:267-270.; Wagner V et al., Acta Allergol 1965;20:1-9.; Yassin A et al., J Laryngol Otol 1974;88:39-46.; Lassalle P et al., Eur J Immunol 1993;23:796-803). On the basis of these studies, previous investigators have suggested that autoimmune mechanism might be involved in the pathogenesis of bronchial asthma and chronic rhinitis. However, previous studies could not establish causal relationship between autoimmunity and asthma due to lack of an identified autoantigen or lack of a logical association between these autoantibodies and airway inflammation. Autoantibody test is not currently used for the diagnosis or classification of bronchial asthma and chronic rhinitis.

Problems in current treatment methods for bronchial asthma and chronic rhinitis Because the primary etiology and mechanism causing the development of bronchial asthma and chronic rhinitis is not completely understood yet, a treatment method that can induce complete remission of bronchial asthma and chronic rhinitis is not developed yet. Current pharmacological therapy can improve the clinical symptoms and physiological functions only during the continuous administration of medication and is not yet proven to modify the long-term natural course of bronchial asthma and chronic rhinitis (National Asthma Education and Prevention Program. NIH publication No. 97- 4051, 1997; Dykewicz MS et al., Ann Allergy Asthma Immunol 1998;81:478-518).

[Detailed Description of the Invention] The present invention is based on the surprising discovery of autoantibodies to cytokeratin 18 protein in serum samples of patients with bronchial asthma and chronic rhinitis, especially in nonallergic patients. The inventors believe this to be the first reporting of such autoantibodies associated with patients with bronchial asthma and chronic rhinitis. Inventors made great efforts to demonstrate the presence of autoantibodies to airway epithelial cell proteins in the bodily fluid of patients with bronchial asthma and chronic rhinitis, and to identify the autoantigen reacting with such autoantibodies. After exhaustive experiments for screening and purification of autoantigen, inventors finally identified the cytokeratin 18 protein is airway epithelial cell autoantigen associated with bronchial asthma and chronic rhinitis, especially in nonallergic patients. The present invention relates to application of cytokeratin 18 protein for the diagnosis and classification of patients with bronchial asthma and chronic rhinitis. The present invention also includes a pharmaceutical formulation comprising cytokeratin 18 protein and methods to protect or treat patients with bronchial asthma and chronic rhinitis using compounds that inhibit the interaction between autoantibodies and cytokeratin 18.

Inventors believe that the autoimmune response against airway epithelial cell protein induce the development of airway inflammation in certain portions of patients with bronchial asthma and chronic rhinitis, especially in nonallergic patients on the basis of following reasons. (1) Airway epithelium has been suggested as being a target for the inflammatory response in bronchial asthma and chronic rhinitis on the basis of pathological studies (Montefort S et al., Clin Exp Allergy 1992; 22:511-520.; Nignola AM et al., Clin Exp Immunol 2001; 31 :674-677.; Wladislavosky-Waserman P et al., Clin Allergy 1984; 14:241-247). (2) Presence of autoantibodies to bronchial mucosa tissue has been reported in patients with bronchial asthma (Girard JP et al., Poumon Coeur 1973; 29:267-270.; Wagner N et al., Acta Allergol 1965;20:1-9) and autoantibodies to nasal mucosa tissue has been reported in patients with chronic rhinitis (Yassin A et al., J Laryngol Otol 1974;88:39-46.). And both bronchial and nasal mucosa tissues are lined by the same type of respiratory epithelial cells.

(3) Analysis of bronchial tissue samples from adult-onset asthmatic patients and from asthmatic patients died of asthmatic attack demonstrated depositions of IgG antibodies and complement in the bronchial epithelium and in the cytoplasm of the bronchial epithelial cells (Molina C et al., Clin Allergy 1977; 7: 137-45; Callerame ML et al., Ν Eng J Med 1971 ; 284: 459-64.).

(4) Autoantibodies to cytokeratin 18 from bodily fluid of patients with bronchial asthma and chronic rhinitis can damage airway epithelial cells through autoantibody- dependent complement-mediated cytotoxicity as disclosed herein.

(5) Removal of plasma containing autoantibodies from a patient with severe asthma induced clinical improvement (Lassalle P et al., Clin Exp Allergy 1990; 20:707-

712.). Intravenous administration of immunoglobulin from healthy donors decreased the requirement of systemic corticosteroid treatment in patients with severe asthma (Salmun LM et al., J Allergy Clin Immunol 1999; 103:810-815.).

Inventors believe that autoantibodies in bodily fluid of patients with bronchial asthma and chronic rhinitis can react with airway epithelial cell protein in upper and lower airway (nasal and bronchial mucosa). And such autoantibody-autoantigen immune complexes can induce chronic inflammation of upper and lower airway by the complement-mediated cytotoxicity and activation of inflammatory cells. And then chronic inflammation of airway can induce the clinical features of bronchial asthma and chronic rhinitis.

The present invention relates to the method for the diagnosis of bronchial asthma and chronic rhinitis, including the steps of (a) obtaining a bodily fluid from a subject suspected of having bronchial asthma and chronic rhinitis (b) contacting the bodily fluid with cytokeratin 18 protein under conditions suitable for the formation of an immune complex between cytokeratin 18 protein and autoantibodies to cytokeratin 18 (c) determining the presence of autoantibodies to cytokeratin 18 by detecting said immune complex, wherein the presence of said immune complex indicates that the subject has bronchial asthma and chronic rhinitis. Techniques to determine the presence of such an immune complex between autoantigen and autoantibodies are known to those skilled in the art, examples of which are disclosed herein. Disclosure of such techniques can be found, for example, in Rose et al., Manual of Clinical Laboratory Immunology, American Society for Microbiology Press, 1997. A bodily fluid can include any fluid collectible from a human subject such as, but not limited to, blood, serum, plasma, urine, tears, saliva, nasal secretion, bronchial secretion, lung secretion, and any other secretions. As used herein, a cytokeratin 18 protein refers to any mammalian cytokeratin 18 or a fragment thereof, such that the fragment retains the ability to bind to the autoantibodies to cytokeratin 18 in bodily fluid from patients with bronchial asthma and chronic rhinitis. A cytokeratin 18 protein can either be isolated or expressed from cells, tissues or microorganisms and can be produced using standard methods in the art, including but not limited to recombinant DNA technology. Human cytokeratin 18 protein consists of 430 amino acids, and the sequence has been reported [reference: Oshima RG, Millan JL, Cecena G. Comparison of mouse and human keratin 18: a component of intermediate filaments expressed prior to implantation. Differentiation 1986; 33: 61-68]. The sequence number 1 (SEQ ID NO: 1) is the amino acid sequence of human cytokeratin 18 protein. Mouse cytokeratin 18 protein consists of 423 amino acids, and the sequence has been reported [Ichinose Y, Morita T, Zhang FY, Srimahasongcram S, Tondella ML, Matsumoto M, Nozaki M, Matsushiro A. Nucleotide sequence and structure of the mouse cytokeratin endoB gene. Gene 1988; 70:85-95]. The sequence number 2 (SEQ ID NO: 2) is the amino acid sequence of mouse cytokeratin 18 protein. Cytokeratin 18 is a cytoskeletal protein found primarily in epithelial cells . lining respiratory and gastrointestinal tracts, including bronchial epithelial cells and lung (alveolar) epithelial cells (Moll R et al., Cell 1982; 31:11-24). Although cytokeratin 18 is a predominantly intracellular protein, its strong expression on the cell surface was also observed in epithelial cells (Moll R et al., Cell 1982; 31:11-24; Saarloos MN et al., Curr Eye Res 1999; 19:439-449).

The present invention includes a method to detect nonallergic patients with bronchial asthma and chronic rhinitis, including steps of (a) obtaining a bodily fluid from a subject suspected of having bronchial asthma and chronic rhinitis (b) contacting the bodily fluid with cytokeratin 18 protein under conditions suitable for the formation of an immune complex between cytokeratin 18 protein and autoantibodies to cytokeratin 18 (c) determining the presence of autoantibodies to cytokeratin 18 by detecting said immune complex, wherein the presence of said immune complex indicates that the subject has nonallergic asthma and rhinitis.

The present invention also includes a method to detect or classify patients with bronchial asthma and chronic rhinitis associated with autoantibodies to cytokeratin 18, including steps of (a) obtaining a bodily fluid from a subject suspected of having bronchial asthma and chronic rhinitis or a patient with bronchial asthma and chronic rhinitis (b) contacting the bodily fluid with cytokeratin 18 protein under conditions suitable for the formation of an immune complex between cytokeratin 18 protein and autoantibodies to cytokeratin 18 (c) determining the presence of autoantibodies to cytokeratin 18 by detecting said immune complex, wherein the presence of said immune complex indicates that the subject or patient has nonallergic asthma and rhinitis associated with autoantibodies to cytokeratin 18.

The present invention also includes a diagnostic kit comprising mammalian cytokeratin 18 protein for detection and classification of the patients with bronchial asthma and chronic rhinitis. Said kit includes mammalian cytokeratin 18 protein or fragments thereof and a means to detect autoantibodies to cytokeratin 18 in bodily fluid from human subjects. The assay methods applied to said kit are known to those skilled in the art, examples of which are disclosed herein. In said kit, assay method includes any techniques that can detect antigen-antibody reaction such as agglutination immunoassays, light-scattering immunoassays, enzyme-linked immunoassays, radioimmunoassays, fluorescence immunoassays, chemiluminescence immunoassays, immunofixation, and immunoblotting but not limited to these.

The present invention also includes a method to prescribe the treatment for bronchial asthma and chronic rhinitis, in that the present invention teaches methods to identify bronchial asthma and chronic rhinitis associated with autoantibodies to cytokeratin 18. Furthermore, the present invention includes a method to monitor the efficacy of a treatment for patients with bronchial asthma and chronic rhinitis, by serially detecting autoantibodies to cytokeratin 18 in the bodily fluid of such patients. The present invention also includes kits comprising mammalian cytokeratin 18 for detection of autoantibodies against cytokeratin 18 to prescribe treatments and monitor the efficacy of treatment in patients with bronchial asthma and chronic rhinitis.

The present invention includes a pharmaceutical formulation comprising mammalian cytokeratin 18 or fragments thereof to protect or treat patients with bronchial asthma and chronic rhinitis who have autoantibodies to cytokeratin 18 in their bodily fluids. Such formulation is designed for the protection of cytokeratin 18-expressing cells from the cytotoxic effect by autoantibodies in bodily fluid of patients with bronchial asthma and chronic rhinitis. The present invention also includes a pharmaceutical formulation comprising a compound that inhibits the binding between autoantibodies to cytokeratin 18 and cytokeratin 18 protein. Such an inhibitory compound includes whole mammalian cytokeratin 18 protein or fragments thereof which retain an ability to bind to autoantibodies against cytokeratin 18.

The present invention includes a method to protect or treat the patients with bronchial asthma and chronic rhinitis who have autoantibodies to cytokeratin 18 protein in their bodily fluids. Such a method includes the step of inhibiting the binding between autoantibodies to cytokeratin 18 and cytokeratin 18-expressing cells by administering an inhibitory compound comprising mammalian cytokeratin 18. Such an inhibitory compound includes whole mammalian cytokeratin 18 protein or fragments thereof retaining an ability to bind to autoantibodies against cytokeratin 18. The present invention also includes a method to identify a pharmaceutical compound capable of inhibiting the binding of autoantibodies from patients with bronchial asthma and chronic rhinitis to cytokeratin 18 protein or cytokeratin 18- expressing cells. Such an inhibitory compound can be identified by the following steps: (a) contacting the autoantibodies isolated from bodily fluid of patients with bronchial asthma and chronic rhinitis with a putative inhibitory compound; and (b) determining whether the compound can inhibit the binding of such autoantibodies to cytokeratin 18 protein or inhibit the cytotoxic effect of autoantibodies to cytokeratin 18-expressing cells.

The details of experiment that identified cytokeratin 18 as an airway epithelial cell autoantigen associated with patients with bronchial asthma and chronic rhinitis are provides as follows.

1. The subjects and methods (1) Subjects

The inventors examined serum samples from 27 patients with allergic asthma and rhinitis, 23 patients with nonallergic asthma and rhinitis, 34 age-matched healthy controls, and 20 patients with systemic lupus erythematosus. All patients with bronchial asthma and chronic rhinitis had typical clinical history compatible with bronchial asthma and chronic rhinitis and documented reversibility of forced expiratory volume in one second

(FEVi) greater than 15% after inhalation of bronchodilator or a 20% decrease in FEVi following the inhalation of less than 8 mg of methacholine/ml. All patients with bronchial asthma and chronic rhinitis underwent skin-prick test with 50 common aeroallergens

(Bencard Co., Brentford, UK). Patients were classified as allergic asthma and rhinitis when wheal diameter of any one allergen was greater than 3 mm over the negative control (normal saline) and there was a definite history or objective evidence of clinical aggravation induced by allergen exposure. Patients with nonallergic asthma and rhinitis showed no positive skin reaction to any of the 50 common aeroallergens and serum total IgE concentrations were within the normal range (less than 180 IU/ml). Twenty patients with systemic lupus erythematosus classified according to the 1982 revised criteria of the American Rheumatic Association were included as control subject with disease. All serum samples from subjects were stored at -20°C.

(2) Culture of airway epithelial cells

Human airway epithelial cell lines including BEAS-2B (ATCC CRL-9609; Ke Y et al., Differentiation 1988; 38:60-6) and A549 (ATCC CCL-185; Giard DJ et al., J Natl Cancer Inst 1973; 51:1417-23) cells were obtained from American Type Culture Collection (ATCC; Manassas, VA).

(3) Cell lysis and protein extraction

Cultured cells were lysed by the addition of lysis buffer containing 10 rαM Tris/HCl, pH 7.2, 1 % Triton X-100, 1% sodium deoxycholate, 0.1% sodium dodecyl sulphate, 158 mM NaCl, 1 mM EGTA, 1 mM Na₃NO₄, 250 μg/ml leupeptin and ImM phenylmethylsulfonyl fluoride.

(4) Immunoblot analysis

Proteins in cell lysates were separated by discontinuous sodium dodecyl sulphate/polyacrylamide gel electrophoresis (SDS-PAGE) using an 8 % resolving gel (pH 8.8) and a 4% stacking gel (pH 6.8). Following electrophoresis, proteins were transferred onto a polyvinylidine difluoride membrane (PNDF; Bio-Rad Laboratories, Hercules, CA). After the transfer, the PNDF membrane was blocked with Tris-buffered saline (TBS) containing 10% bovine serum and 0.1% Tween 20. The PNDF membrane strips were then probed with 1 ml of serum samples at 1 in 100 dilution for 2 hours at room temperature. After washes, the membrane was incubated with alkaline phosphates-conjugated goat anti-human IgG (Sigma Chemical Co., St. Louis, MO) for 2 hours at room temperature. After a final washing, the membrane was stained with a BCIP/ΝBT solution (5-bromo-4-chloro-3-indoyl phosphate/nitro blue tetrazolium; Sigma). To confirm the identification of 49-kDa autoantigen after the amino acid sequence analysis, a mouse monoclonal antibody to human cytokeratin 18 (clone no. CY- 90, Sigma) and a negative control mouse monoclonal antibody with the same IgGl isotype (Sigma Chemical Co., St. Louis, MO) were used for immunoblot analysis. Alkaline phosphatase-conjugated goat anti-mouse IgG (Sigma Chemical Co., St. Louis, MO) was used as secondary conjugate and the results were developed as above. Commercially available purified bovine cytokeratin 18 protein (Research Diagnostics INC., Pleasant Hill Road Flanders, NJ) was used as a positive control antigen in the experiment using mouse monoclonal antibody to human cytokeratin 18.

(5) Purification and identification of autoantigen

For purification of the autoantigen, airway epithelial cell (BEAS-2B) lysates were fractionated by ion-exchange chromatography with diethylaminoethyl (DEAE) Sepharose bead (Sigma Chemical Co., St. Louis, MO). Fractions of interest were analyzed by SDS-PAGE and immunoblot analysis and further concentrated with Centriprep-50 (Amicon, Witten, Germany) and subjected to reverse-phase high-performance liquid chromatography (HPLC) using Vydac C18 column (The Separation Group, Inc., Hesperia, CA). Fractions were collected and lyophilized. They were examined by SDS-PAGE and immunoblot analysis. Because analysis of purified protein on PVDF revealed that the N- terminal amino acid sequence was blocked, the protein was subjected to enzymatic in-gel digestion by trypsin. Trypsin-digested peptide fragments were separated by micro- HPLC system using Sephasil C18 reverse-phase column (Amersham Pharmacia Biotech, Uppsala, Sweden). Two fractions of peptide fragments were subjected to amino acid sequencing using Procise cLC 492 Protein sequencing system (Applied Biosystems, Foster, CA). To compare the amino acid sequences of peptide fragments with known protein sequences, the SWISS-PROT database (Swiss Institute of Bioinfromatics, Geneva, Switzerland; The European Bioinfromatics Institute, Cambridge, U.K.) was used.

(6) Detection of other autoantibodies All the serum samples were also tested for IgG antinuclear antibodies (ANA), and

IgG autoantibodies to thyroglobulin and thyroid peroxidase. IgG antinuclear antibodies were assessed by an indirect immunofluorescence staining of HEp2 cell (Hemagen Diagnostics Inc., Maryland, USA), and IgG autoantibodies to thyroglobulin and thyroid peroxidase were measured by radioimmunoassay (BRAHMS DIAGNOSTICA GMBH, Berlin, Germany).

(7) Complement-mediated cytotoxicity to airway epithelial cell by autoantibodies The complement-mediated cytotoxicity to airway epithelial cell by autoantibodies was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). The experiment was conducted using serum samples from 8 patients with nonallergic asthma and rhinitis who have IgG autoantibodies to cytokeratin 18, 6 patients with nonallergic asthma and rhinitis who have no detectable IgG autoantibodies to cytokeratin 18, 8 patients with allergic asthma and rhinitis who have no detectable IgG autoantibodies to cytokeratin 18, and 8 healthy controls. The airway epithelial cell (BEAS-2B) was cultured on the 96 well culture plate. When the cells covered 50% surface of each well, the cytotoxicity was measured. Before the measurement of cytotoxicity, the serum samples were treated at 56 ^°C for 30 minutes to inactivate the complement. After the culture medium was removed from each well, 100 of triplicated serum samples diluted 1 :20 in DMEM/F12 medium were added to each well or DMEM/F12 medium only was added to each well as a control. The plate was incubated for 90 minutes at 37^°C in the CO₂ incubator. Then, the 10/ of rabbit complement was added to each well and the mixture incubated for 90 minutes at 37^°C in the CO incubator. Culture medium including serum sample and complement was removed from each well and 200/^ of fresh DMEM/F12 medium including 10% fetal bovine serum and 50 ii of the MTT solution was added to each well and the plate was incubated for 3 hours at 37 "Cin the CO₂ incubator. Then the supernatant was removed from each well and 200μi of dimethyl sulfoxide was added to each well. And then 25 ti of 0.1 M glycine, 0.1 M NaCl buffer (pH 10.5) was added to each well. Absorbance at 570 run was measured by microplate reader. Complement-mediated cytotoxicity was expressed as % cell lysis using following formula with absorbance values of test wells including serum samples and control wells including the culture medium only. Cytotoxicity (% cell lysis) = [(absorbance of control wells - absorbance of test wells)/ absorbance of control wells] X 100.

(8) Inhibition of complement-mediated cytotoxicity to airway epithelial cell

The same volumes of serum samples from 8 patients with nonallergic asthma and rhinitis who have IgG autoantibodies to cytokeratin 18 were mixed and made into a pooled serum sample. This pooled serum was treated for 30 minutes at 56 ^°C and diluted 1 :20 in DMEM/F12 medium and the complement-mediated airway epithelial cytotoxicity was measured. To inhibit a complement-mediated cytotoxicity to airway epithelial cell in the pooled serum, purified human cytokeratin 18 protein or human serum albumin was added to the diluted pooled serum at the lOOμg/mβ final concentration of inhibitors. The mixture was incubated for 2 hours at 37C and then complement-mediated cytotoxicity was measured. The results were obtained from 8 individual experiments and expressed as mean and standard deviation.

2. The results

(1) Detection of IgG autoantibodies to bronchial epithelial cell antigen

IgG autoantibodies to 49-kDa bronchial epithelial cell antigen were detected in serum samples from 10 of 23 patients with nonallergic asthma and rhinitis (43%), 3 of 27 patients with allergic asthma and rhinitis (11%), 2 of 20 patients with systemic lupus erythematosus (10%), and 3 of 34 age-matched healthy controls (9%) (Figure 1, Table 1; chi-square test, p<0.005). The positive rate of IgG autoantibodies to 49-kDa bronchial epithelial cell antigen was significantly higher in patients with nonallergic asthma and rhinitis compared to patients with allergic asthma and rhinitis, patients with systemic lupus erythematosus, and healthy controls (Table 1 ; Fisher's exact test, p<0.05).

Table 1 shows the detection rate of IgG autoantibodies to the 49-kDa airway epithelial cell antigens in patients with allergic asthma and rhinitis, patients with nonallergic asthma and rhinitis, patients with systemic lupus erythematosus, and the healthy controls.

< Table 1>

*A statistical significance of the difference between two groups (nonallergic asthma and rhinitis versus other group) was calculated by the Fisher's exact test.

(2) Detection of other IgG autoantibodies The positive rates of IgG antinuclear antibodies and IgG autoantibodies to thyroid autoantigens were not significantly different among patients with nonallergic asthma and rhinitis, patients with allergic asthma and rhinitis, and healthy controls (Table 2, p>0.05).

Table 2 shows the detection rate of IgG antinuclear antibodies, IgG autoantibodies to thyroglobulin, and IgG autoantibodies to thyroid peroxidase in patients with allergic asthma and rhinitis, patients with nonallergic asthma and rhinitis, patients with systemic lupus erythematosus, and healthy controls.

< Table 2>

Systemic lupus erythematosus 20 19 (95%)* 3 (15%) 3 (15%)

ANA: antinuclear antibodies, Anti-TG: anti-thyroglobulin antibodies, Anti-TPO: anti-thyroid peroxidase antibodies. *A significant statistical difference compared with 3 other groups (p<0.05).

(3) Purification and identification of 49-kDa airway epithelial cell autoantigen

To characterize the 49-kDa airway epithelial cell autoantigen, this protein was purified by ion-exchange chromatography and reverse-phase HPLC. The purified protein was separated in an 8% tris-glycine gel (Figure 2). The purified protein was then subjected to enzymatic in-gel digestion by trypsin, and the peptide fragments were separated by reverse-phase HPLC (Figure 3). The two fractions (peak A and peak B) of peptide fragments were subjected to amino acid sequencing. Amino acid sequences of two peptide fragments were found completely compatible with human cytokeratin 18 protein upon database analysis (Table 3).

Table 3 shows the amino acid sequences of two peptide fragments of the purified

49-kDa autoantigen and compatible amino acid sequences in the database. < Table 3>

The identification of the 49-kDa airway epithelial cell autoantigen as human cytokeratin 18 was further confirmed by immunoblot analysis using monoplonal antibody against human cytokeratin 18 and comparing with purified bovine cytokeratin 18 protein (Figure 4).

(4) Complement-mediated cytotoxicity to airway epithelial cell by autoantibodies Complement-mediated cytotoxicity to airway epithelial cell was significantly higher in the serum samples of patients with nonallergic asthma and rhinitis who have

IgG autoantibodies to cytokeratin 18 (mean ± standard deviation; 30.9 ± 10.2%) than patients with nonallergic asthma and rhinitis who have no detectable IgG autoantibodies to cytokeratin 18 (19.1 ± 3.1%), patients with allergic asthma and rhinitis who have no detectable IgG autoantibodies to cytokeratin 18 (16.5 ± 2.7%), and healthy controls (15.8 ± 3.8%) (Figure 8, p<0.005). Complement-mediated cytotoxicity to airway epithelial cell was not detectable when the heat-inactivated serum samples were only added without complement. Moreover, complement-mediated cytotoxicity to airway epithelial cell in the pooled serum sample (29.1 ± 4.3%) was significantly inhibited by addition of the purified human cytokeratin 18 protein (11.3 ± 2.6%) but not by addition of human serum albumin (27.3 ± 2.7%) (Figure 9, p<0.005). These results demonstrate that airway epithelial cell can be damaged by autoantibodies to cytokeratin 18 existing in the serum samples of patients with nonallergic asthma and rhinitis through the complement-mediated cytotoxicity. And a significant inhibition of complement-mediated cytotoxicity to airway epithelial cells by purified human cytokeratin 18 protein clearly demonstrates that human cytokeratin 18 protein can protect the airway epithelial cell from the damage by autoantibodies in bodily fluid from patients with bronchial asthma and chronic rhinitis who have autoantibodies to cytokeratin 18 protein. And this result also indicates that administration of human cytokeratin 18 protein can protect the patients with bronchial asthma and chronic rhinitis who have autoantibodies to cytokeratin 18 from the airway epithelial cell damage by circulating autoantibodies.

[Brief description of the drawings ]

Figure 1 shows immunoblot analysis of IgG autoantibodies to human airway epithelial cell (BEAS-2B) antigens in serum samples from healthy controls (lane 1-3), patients with allergic asthma and rhinitis (lane 4-7), patients with nonallergic asthma and rhinitis (lane 8-11), patients with systemic lupus erythematosus (lane 12-14), a patient with nonallergic asthma and rhinitis as a positive control (lane 15), and dilution buffer only as a negative control (lane 16). * Arrow indicates the 49-kDa autoantigen.

Figure 2 shows the protein staining of purified 49-kDa autoantigen separated by

SDS-PAGE. Protein staining shows molecular weight standard (lane 1), whole cell extract of airway epithelial cell (BEAS-2B) (lane 2), 49-kDa autoantigen purified by ion- exchange chromatography and reverse-phase HPLC (lane 3), and purified bovine cytokeratin 18 protein (lane 4).

Figure 3 shows the chromatograph of peptide fragments derived from trypsin- digestion of purified 49-kDa autoantigen separated by reverse-phase HPLC. Two fractions of peptide fragments (peaks A and B) were sequenced. Figure 4 shows immunoblot analysis of IgG autoantibodies in serum samples from two patients with nonallergic asthma and rhinitis and a monoclonal antibody to cytokeratin 18. Whole cell extract of airway epithelial cell (BEAS-2B) (lanes 1, 4, and 7), purified 49-kDa autoantigen (lanes 2, 5, and 8), and purified bovine cytokeratin 18 (lanes 3, 6, and 9) were subjected to immunoblot analysis. Autoantibodies in serum samples from two patients with nonallergic asthma and rhinitis (lanes 1-3, lane 7-9) and monoclonal antibody to cytokeratin 18 (lanes 4-6) recognized the purified 49-kDa autoantigen and purified bovine cytokeratin 18.

Figure 5 shows immunoblot analysis of IgG autoantibodies to human cytokeratin 18 protein in serum samples from healthy controls (lane 1, 2), a patient with allergic asthma and rhinitis (lane 3), patients with nonallergic asthma and rhinitis (lane 4-6). A monoclonal antibody to cytokeratin 18 was used as a positive control (lane 7) and dilution buffer only was used as a negative control (lane 8). * Arrow indicates the cytokeratin 18 protein.

Figure 6 shows detection of IgG autoantibodies to purified human cytokeratin 18 protein in serum samples from 2 patients with nonallergic asthma and rhinitis and a pooled serum of 10 healthy controls by enzyme-linked immunosorbent assay (ELISA).

Figure 7 shows immunoblot analysis of IgA autoantibodies to cytokeratin 18 protein in serum samples from a healthy control (lane 1), patients with allergic asthma and rhinitis (lane 2-4), and patients with nonallergic asthma and rhinitis (lane 5-10). A monoclonal antibody to cytokeratin 18 was used as a positive control (lane 11). * Arrow indicates the cytokeratin 18 protein.

Figure 8 shows complement-mediated cytotoxicity to airway epithelial cell (BEAS-2B) in serum samples from healthy controls (group 1), patients with allergic asthma and rhinitis who have no detectable IgG autoantibodies to cytokeratin 18 (group 2), patients with nonallergic asthma and rhinitis who have no detectable IgG autoantibodies to cytokeratin 18 (group 3), and patients with nonallergic asthma and rhinitis who have IgG autoantibodies to cytokeratin 18 (group 4).

Figure 9 shows complement-mediated cytotoxicity to airway epithelial cell (BEAS-2B) in the pooled serum sample of patients who have IgG autoantibodies to cytokeratin 18 (Serum only) and inhibition of the cytotoxicity by addition of purified human cytokeratin 18 protein (Serum + CK18) or human serum albumin (Serum + HSA). The data were obtained from 8 individual experiments and expressed as mean and standard deviation.

Figure 10 shows immunoblot analysis of IgG autoantibodies to purified human cytokeratin 18 protein in serum samples of allergic asthmatic patients without clinical evidence of chronic rhinitis (lane 1 -3) and nonallergic asthmatic patients without clinical evidence of chronic rhinitis (lane 4-7). A monoclonal antibody to cytokeratin 18 was used as a positive control (lane 8). * Arrow indicates the cytokeratin 18 protein.

Figure 11 shows immunoblot analysis of IgG autoantibodies to cytokeratin 18 protein in serum samples of allergic rhinitis patients without bronchial asthma (lane 1-3), nonallergic rhinitis patients without bronchial asthma (lane 4-7), and healthy controls

(lane 9, 10). A monoclonal antibody to cytokeratin 18 was used as a positive control (lane

8). * Arrow indicates the cytokeratin 18 protein.

The following embodiments are provided for the purpose of illustration and are not intended to limit the scope of the present invention.

[Detailed Description of the Preferred Embodiments] The embodiment 1 Detection of IgG and IgA autoantibodies to cytokeratin 18 in serum samples from patients with bronchial asthma and chronic rhinitis by immunoblot analysis.

Whole cell extract from human airway epithelial cells (A549 cell) or purified human cytokeratin 18 protein was separated by SDS-PAGE (4% stacking gel and 8% running gel), protein was transferred onto the PVDF membrane. The PNDF membrane was incubated with TBS containing 5% nonfat dried milk and 0.05% Tween 20 (blocking buffer) for 1 hour to prevent the nonspecific protein bindings to PVDF, the membrane was made to 4_Q_α-width strips. The PVDF strips were incubated with serum samples diluted 1 :100 in blocking buffer for 2 hours at room temperature. After washing, the PVDF strips were incubated with alkaline phosphatase-conjugated goat anti-human IgG or anti-human IgA antibodies for 2 hours. After washing, the PVDF strips were stained with BCIP/ΝBT substrate solution for 5 minutes. As a positive control, one PNDF strip was incubated with mouse monoclonal antibody to cytokeratin 18 instead of human serum sample and alkaline phosphatase-conjugated goat anti-mouse IgG antibodies were used as secondary conjugate and stained by BCIP/ΝBT. As a negative control, one PVDF strip was incubated with blocking buffer only instead of serum samples. On the immunoblot analysis of IgG autoantibodies to cytokeratin 18 in serum samples using whole cell extract of human airway epithelial cells (A549), IgG autoantibody to cytokeratin 18 was negative in 2 healthy controls and a patient with allergic asthma and rhinitis and positive in 3 patients with nonallergic asthma and rhinitis (Figure 5). On the immunoblot analysis of IgA autoantibodies to cytokeratin 18 in serum samples using whole cell extract of human airway epithelial cells (A549), IgA autoantibody to cytokeratin 18 was negative in a healthy control and 3 patients with allergic asthma and rhinitis and positive in 6 patients with nonallergic asthma and rhinitis (Figure 7). On the immunoblot analysis of IgG autoantibodies to cytokeratin 18 in serum samples using purified human cytokeratin 18 protein, IgG autoantibody to cytokeratin 18 was negative in 3 allergic asthmatic patients without clinical evidence of chronic rhinitis and positive in 4 nonallergic asthmatic patients without clinical evidence of chronic rhinitis (Figure 10). On the immunoblot analysis of IgG autoantibodies to cytokeratin 18 in serum samples using whole cell extract of human airway epithelial cells (A549), IgG autoantibody to cytokeratin 18 was negative in 2 healthy controls and 3 allergic rhinitis patients without bronchial asthma and positive in 4 nonallergic rhinitis patients without bronchial asthma (Figure 11).

The embodiment 2

Detection of IgG autoantibodies to cytokeratin 18 in serum samples from patients with bronchial asthma and chronic rhinitis by enzyme-linked immunosorbent assay (ELISA) Microtiter plates were coated with purified human cytokeratin 18 protein at a o concentration of 0.5 μg per well in 0.1M carbonate buffer (pH 9.6) for 16 hours at 4 C. After washing 3 times with phosphate buffered saline containing 0.05% Tween-20 (PBST), wells were incubated with 350μl of PBST containing 3% fetal bovine serum for 1 hour at room temperature. After washing 3 times with PBST, wells were incubated with 100 μl of quadruplicated serum samples diluted in PBST containing 3% fetal bovine serum for 2 hours. After washing 3 times, wells were incubated with peroxidase- conjugated goat anti-human IgG antibodies (Sigma) for 2 hours. After washing 3 times, 100 μl of the TMB substrate solution (Sigma) was added to each well. After 10 minutes, the reaction was stopped by adding 100 μl of 2.5 N H₂SO₄to each well. The absorbance was measured at 450 nm using ELISA reader. Absorbance values from serum samples of 2 patients with nonallergic asthma and rhinitis were significantly higher than the absorbance values from a pooled serum sample of 10 healthy controls (Figure 6).

The embodiment 3 Method to prescribe treatment for bronchial asthma by detection of IgG autoantibodies to cytokeratin 18 in the serum samples

Although several non-steroidal immunomodulatory drugs such as intravenous immunoglobulin, cyclosporine, gold, methotrexate, and hydroxychloroquine have been reported to be beneficial to severe asthmatic patients, their use in asthma remains complicated because of highly variable effects in individual patients and the absence of marker predicting responsiveness to such treatments.

Here present invention shows a method to prescribe intravenous immunoglobulin for patients with severe asthma on the basis of detection of IgG autoantibodies to cytokeratin 18 in the serum samples. Two adult patients with nonallergic asthma and rhinitis were admitted to hospital due to severe aggravation of their asthmatic symptoms. And the two patients received standard therapy for exacerbation of asthma including high dose intravenous corticosteroid therapy (62.5mg of methyl prednisolone per 6 hours) and maximal doses of nebulized bronchodilator (salbutamol) with nasal oxygen supply for 5 days. However, their asthmatic symptoms and pulmonary functions were not improved. After informed consent, high dose of intravenous immunoglobulin (0.4g/kg/day) was administered to the two patients for 2 days (admission day 6, 7) with continuation of standard therapy. After intravenous immunoglobulin therapy, patient 1 showed dramatic clinical improvement of asthmatic symptoms and objective pulmonary function parameter but patient 2 did not show significant improvement (Table 4).

Table 4 shows changes of asthma severity in two patients with nonallergic asthma and rhinitis who admitted to hospital because of asthma exacerbation. Asthma severity was expressed as peak expiratory flow rate (PEFR) that was the mean value of 3 -time measurements at 7:00 am before the use of inhaled bronchodilator. <Table 4>

"Intravenous immunoglobulin (0.4g kg/day) was administered to patients at admission day 6 and 7.

Immunoblot detection of IgG autoantibodies to cytokeratin 18 in serum samples taken at day 1 showed positive result in patient 1 (Figure 4, lanes 1-3) and negative result in patient 2. These results indicate that detection of IgG autoantibodies to cytokeratin 18 in serum samples from patients with bronchial asthma and chronic rhinitis can be used as a marker predicting responsiveness to immunomodulatory treatment including intravenous immunoglobulin therapy.

[Industrial Applicability] The present invention can be used for screening of patients with bronchial asthma and chronic rhinitis by a simple blood test detecting autoantibodies to cytokeratin 18 instead of complex steps of clinical evaluation and laboratory tests. Present invention also can be used for the detection of nonallergic patients with bronchial asthma and chronic rhinitis showing autoimmune phenomenon by detecting autoantibodies to cytokeratin 18. And present invention can be used for the classification of patients with bronchial asthma and chronic rhinitis showing autoimmune phenomenon by detecting autoantibodies to cytokeratin 18. The present invention can be used to prescribe a specific treatment for patients with bronchial asthma and chronic rhinitis by detecting autoantibodies to cytokeratin 18. The present invention can be used for a pharmaceutical formulation comprising cytokeratin 18 protein or fragments thereof to protect the patients with bronchial asthma and chronic rhinitis associated with autoantibodies to cytokeratin 18. The present invention also can be used to identify a pharmaceutical compound capable of inhibiting the binding ability of autoantibodies to cytokeratin 18 from patients with bronchial asthma and chronic rhinitis to cytokeratin 18 protein or cytokeratin 18-expressing cells.

Claims

What is claimed is:

1. A method for diagnosing bronchial asthma and chronic rhinitis, said method comprising detecting autoantibodies to cytokeratin 18 protein in the bodily fluid of a human subject.

2. A method to detect nonallergic patients with bronchial asthma and chronic rhinitis, said method comprising detecting autoantibodies to cytokeratin 18 protein in the bodily fluid of a human subject.

3. A method to detect patients with bronchial asthma and rhinitis associated with autoantibodies to cytokeratin 18 protein, said method comprising detecting autoantibodies to cytokeratin 18 protein in the bodily fluid of a human subject.

4. A method to prescribe a treatment for bronchial asthma and chronic rhinitis, said method comprising: (a) detection of autoantibodies to cytokeratin 18 protein in the bodily fluid of a subject; and (b) prescribing a treatment for bronchial asthma and chronic rhinitis as identified in step (a).

5. A method for monitoring the efficacy of a treatment for bronchial asthma and chronic rhinitis, said method comprising detection of autoantibodies to cytokeratin 18 protein in the bodily fluid of human subject.

6. The method as claimed in any one of claims 1 to 5, wherein said method comprises the following steps of: (a) obtaining a bodily fluid from a subject (b) contacting the bodily fluid with cytokeratin 18 protein under conditions suitable for the formation of an immune complex between cytokeratin 18 protein and autoantibodies to cytokeratin 18 (c) determining the presence of autoantibodies to cytokeratin 18 by detecting said immune complex, presence of said immune complex indicating the presence of autoantibodies to cytokeratin 18 in said subject.

7. The method as claimed in any one of claims 1 to 6, wherein said cytokeratin 18 protein is mammalian.

8. The method as claimed in any one of claims 1 to 6, wherein said cytokeratin 18 protein has the amino acid sequence of SEQ ID NO: 1, or a modification or fragment thereof having immunological reactivity with autoantibodies to cytokeratin 18.

9. The method as claimed in any one of claims 1 to 6, wherein said cytokeratin 18 protein has the amino acid sequence of SEQ ID NO: 2, or a modification or fragment thereof having immunological reactivity with autoantibodies to cytokeratin 18.

10. The method as claimed in any one of claims 1 to 6, wherein said cytokeratin 18 protein is a fragment of cytokeratin 18 protein retaining an ability to interact with autoantibodies to cytokeratin 18.

11. A kit for diagnosing bronchial asthma and chronic rhinitis, said kit comprising cytokeratin 18 protein and a means to detect autoantibodies to cytokeratin 18 in the bodily fluid of a human subject.

12. A kit to detect nonallergic patients with bronchial asthma and chronic rhinitis, said kit comprising cytokeratin 18 protein and a means to detect autoantibodies to cytokeratin 18 in the bodily fluid of a human subject.

13. A kit to detect patients with bronchial asthma and rhinitis associated with autoantibodies to cytokeratin 18, said kit comprising cytokeratin 18 protein and a means to detect autoantibodies to cytokeratin 18 in the bodily fluid of a human subject.

14. A kit to prescribe treatment for bronchial asthma and chronic rhinitis, said kit comprising cytokeratin 18 protein and a means to detect autoantibodies to cytokeratin 18 in the bodily fluid of a human subject.

15. A kit for monitoring the efficacy of a treatment for bronchial asthma and chronic rhinitis, said kit comprising cytokeratin 18 protein and a means to detect autoantibodies to cytokeratin 18 in the bodily fluid of a human subject.

16. The kit as claimed in any one of claims 11 to 15, wherein said kit comprising (a) cytokeratin 18 protein (b) reagents for preparing a medium for an immunological reaction (c) reagents capable of detecting an immune complex of cytokeratin 18 protein and autoantibodies to cytokeratin 18.

17. The kit as claimed in any one of claims 11 to 16, wherein said cytokeratin 18 protein is mammalian.

18. The kit as claimed in any one of claims 11 to 16, wherein said cytokeratin 18 protein has the amino acid sequence of SEQ ID NO: 1, or a modification or fragment thereof having immunological reactivity with autoantibodies to cytokeratin 18.

19. The kit as claimed in any one of claims 11 to 16, wherein said cytokeratin 18 protein has the amino acid sequence of SEQ ID NO: 2, or a modification or fragment thereof having immunological reactivity with autoantibodies to cytokeratin 18.

20. The kit as claimed in any one of claims 11 to 16, wherein said cytokeratin 18 protein is a fragment of cytokeratin 18 protein retaining an ability to interact with autoantibodies to cytokeratin 18.

21. A pharmaceutical formulation comprising cytokeratin 18 protein for patients with bronchial asthma and chronic rhinitis.

22. A pharmaceutical formulation comprising cytokeratin 18 protein for nonallergic patients with bronchial asthma and chronic rhinitis.

23. A pharmaceutical formulation comprising cytokeratin 18 protein for patients with bronchial asthma and chronic rhinitis associated with autoantibodies to cytokeratin 18 protein.

24. A pharmaceutical formulation comprising cytokeratin 18 protein to protect or treat patients with bronchial asthma and chronic rhinitis.

25. A pharmaceutical formulation comprising cytokeratin 18 protein to protect or treat nonallergic patients with bronchial asthma and chronic rhinitis.

26. A pharmaceutical formulation comprising cytokeratin 18 protein to protect or treat patients with bronchial asthma and chronic rhinitis associated with autoantibodies to cytokeratin 18 protein.

27. The inventions of claim 21 to 26, wherein said cytokeratin 18 protein is mammalian.

28. The inventions of claim 21 to 26, wherein said cytokeratin 18 protein has the amino acid sequence of SEQ ID NO: 1, or a modification or fragment thereof having immunological reactivity with autoantibodies to cytokeratin 18.

29. The inventions of claim 21 to 26, wherein said cytokeratin 18 protein has the amino acid sequence of SEQ ID NO: 2, or a modification or fragment thereof having immunological reactivity with autoantibodies to cytokeratin 18.

30. The inventions of claim 21 to 26, wherein said cytokeratin 18 protein is a fragment of cytokeratin 18 protein retaining the ability to interact with autoantibodies to cytokeratin 18.

31. A pharmaceutical formulation comprising compounds capable of inhibiting binding between cytokeratin 18 protein and autoantibodies to cytokeratin 18 from patients with bronchial asthma and chronic rhinitis.

32. A pharmaceutical formulation comprising compounds capable of inhibiting the cytotoxic effect of autoantibodies from patients with bronchial asthma and chronic rhinitis to cytokeratin 18-expressing cells.

33. A method to identify a pharmaceutical compound capable of inhibiting binding between cytokeratin 18 protein and autoantibodies to cytokeratin 18 from patients with bronchial asthma and chronic rhinitis.

34. A method to identify pharmaceutical compounds inhibiting the cytotoxic effect of autoantibodies from patients with bronchial asthma and chronic rhinitis to cytokeratin 18-expressing cells.

35. The invention of claims 31 to 34, wherein said pharmaceutical compounds are formulated to protect or treat patients with bronchial asthma and chronic rhinitis.

36. The invention of claims 31 to 34, wherein said pharmaceutical compounds are formulated to protect or treat nonallergic patients with bronchial asthma and chronic rhinitis.

37. The invention of claims 31 to 34, wherein said pharmaceutical compounds are formulated for patients with bronchial asthma and chronic rhinitis associated with autoantibodies to cytokeratin 18.

38. A method to protect or treat patients with bronchial asthma and chronic rhinitis by administering a pharmaceutical formulation comprising cytokeratin 18 protein.

39. A method to protect or treat nonallergic patients with bronchial asthma and chronic rhinitis by administering a pharmaceutical formulation comprising cytokeratin 18 protein.

40. A method to protect or treat patients with bronchial asthma and chronic rhinitis associated with autoantibodies to cytokeratin 18 by administering a pharmaceutical formulation comprising cytokeratin 18 protein.

41. The invention of claims 38 to 40, wherein said cytokeratin 18 is mammalian.

42. The invention of claims 38 to 40, wherein said cytokeratin 18 has the amino acid sequence of SEQ ID NO: 1, or a modification or fragment thereof having immunological reactivity with autoantibodies to cytokeratin 18.

43. The invention of claims 38 to 40, wherein said cytokeratin 18 has the amino acid sequence of SEQ ID NO: 2, or a modification or fragment thereof having immunological reactivity with autoantibodies to cytokeratin 18.

44. The invention of claims 38 to 40, wherein said cytokeratin 18 is a fragment of cytokeratin 18 protein retaining an ability to interact with autoantibodies to cytokeratin 18.

45. A method to protect or treat patients with bronchial asthma and chronic rhinitis by administering a pharmaceutical compound capable of inhibiting binding between cytokeratin 18 protein and autoantibodies to cytokeratin 18 from patients with bronchial asthma and chronic rhinitis.

46. A method to protect or treat nonallergic patients with bronchial asthma and chronic rhinitis by administering a pharmaceutical compound capable of inhibiting binding between cytokeratin 18 protein and autoantibodies to cytokeratin 18 from patients with bronchial asthma and chronic rhinitis.

47. A method to protect or treat patients with bronchial asthma and chronic rhinitis associated with autoantibodies to cytokeratin 18 by administering a pharmaceutical compound capable of inhibiting binding between cytokeratin 18 protein and autoantibodies to cytokeratin 18 from patients with bronchial asthma and chronic rhinitis.

48. Use of cytokeratin 18 protein for diagnosing bronchial asthma and chronic rhinitis.

49. Use of cytokeratin 18 protein for classifying bronchial asthma and chronic rhinitis.

50. Use of cytokeratin 18 protein for pharmaceutical formulation to protect or treat patients with bronchial asthma and chronic rhinitis.

51. Use of cytokeratin 18 protein as a drug target in the production of drugs for the treatment of bronchial asthma and chronic rhinitis.

52. Use of cytokeratin 18 protein for the treatment of patients with bronchial asthma and chronic rhinitis.